HAL Id: hal-02779339
https://hal.inrae.fr/hal-02779339
Submitted on 4 Jun 2020
HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.
L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.
In vitro estrogenic activity of phytoestrogens on liver vitellogenin synthesis in the rainbow trout
(Oncorhynchus mykiss)
C. Pelissero, Jean-Luc Foucher, B. Bennetau, J. Dunogues, G. Flouriot, J.P.
Sumpter
To cite this version:
C. Pelissero, Jean-Luc Foucher, B. Bennetau, J. Dunogues, G. Flouriot, et al.. In vitro estrogenic activity of phytoestrogens on liver vitellogenin synthesis in the rainbow trout (Oncorhynchus mykiss).
4. International Symposium on the Reproductive Physiology of Fish, Jul 1991, Norwich, United Kingdom. 338 p. �hal-02779339�
In_yllm ESTROGENIC ACTIVITY OF PHYTOESTROGENS ON LIVER VITELLOGENIN SYNTHESIS IN THE RAINBOW TROUT (Oncorhvnchus mykjss).
C. Pelissero 1, J. L. Foucher 2, B. Bennetau 3, J. Dunoguès 3, G. Flouriot 4 and J. P. Sumpter 1.
1. Department of Biology and Biochemistry, Brunel University, Uxbridge Middlesex, UB8 3PH, U. K.
2. Laboratoire INRA de Physiologie des Poissons, Université de Rennes I, 35042 Rennes cedex, France.
3. URA 35 CNRS, Université de Bordeaux 1, 351 Cours de la Libération, 33405 Talence cedex, France.
4. Laboratoire de Biologie Moléculaire, Université de Rennes 1, 35042 Rennes cedex, France.
Summarv
Phytoestrogens from vegetable sources Iike soya or alfalfa have been demonstrated to be patent in vivo in stimulating vitellogenin (VTG) secretion in cultured Siberian sturgeon (Pelissero ~. 1991a, 1991b).
These phytoestrogens can be received via the diet. In Sturgeon, a commercial diet can spontaneously induce vitellogenin secretion in both males and females, suggesting a high sensitivity of this species to the action of the phytoestrogens. Based on these results, we tested the major phytoestrogens for their capacity to stimulate cultured trout hepatocytes to synthesize and secrete vitellogenin.The results shown here demontrate the reliability of the cell culture technique as well as its use to detennine the in vitro potency of phytoestrogens on VTG secretion. According to our results, all the phytoestrogens are 1000 to 2000 times less patent than 17 B-estradiol.
Introduction
When trying to sex Siberian sturgeon, Acipencer Q.Kri, by measuring their plasma VTG Ievels we surprisingly found it in plasma of mature and immature fish of both sexes (Pelissero ~. 1989). This result was unexpected, since vitellogenin is normally synthesized only by female fish during oocyte development (Copeland ~. 1986). The discovery of starved contrai fish which had no detectable VTG in the plasma led us to consider that our fish could be subject to dietary estrogenic contamination. Based on the data already available in the literature, we discovered that estrogenic compounds called phytoestrogens were present at high concentrations (several mg/lOOg) in vegetable sources like soya (Setchell, 1985) or alfalfa (Knukles ~. 1976) and the main products manufactured from them. Since soya and alfalfa are often present in high amount in commercial fish diets, we decided to test on sturgeon both the estrogenic effect of a soya based diet, and a commercial diet and compare them to a control diet, free of estrogenic compounds, based on casein. The results obtained have been presented in Pelissero kL.l!L.
1991b and they demonstrate the estrogenic potency of both the soya based diet and the commercial diet on immature Siberian sturgeon.
Phytoestrogens were then synthesized and tested in YiY2 by intraperitoneal injection into yearling Siberian
sturgeon (Pelissero tl..!!L, 199la). Although the high cost of the phytotestrogens meant only a single dose of each was tested, the results nevertheless clearly demonstrated that many of the phytoestrogens induced VTG synthesis. However, the relative potencies of the various phytoestrogens could not be detennined from the experiment conducted.
In order to learn more about the estrogenicity of phytoestrogens, we have developped a bioassay based upon the quantification of vitellogenin secreted into the medium from cultured hepatocytes of rainbow trout.
The work presented here has allowed us to assess the estrogenic potency of the various phytoestrogens.
Materia! and Methods
Male, immature female or sterile trout were collected Crom a local fish farm. Their weight varied from 350g to 800g. They were kept in an aquarium at Brunel University for at least two weeks before the experiment
The cell culture technique was first developped by G. Flouriot in the Molecular Biology Laboratory of the University of Rennes I, modified by J. L. Foucher Crom the INRA Fish Physiology Laboratory of Rennes and then adapted to our purpose. The cells were obtained by perfusion of the liver for 30 min with 300 ml Hepes buffered saline containing 230 mg/!
collagenase (Boeringher Mannheim U. K.). The cell suspension obtained was adjusted to a concentration of 2 to 4 x106 cells / ml in Hepes buffered (pH 7.8) Dulbecco's modified Eagle's medium (DMEM;
Sigma). It was then distributed in 6 well-culture dishes (0 3.5 cm; NUNC) using 3 ml/ well. The plates were incubated at 18°C on a shaker with the rotation speed set at 20 revolutions / min. The cells were incubated for 4 days without any stimulation, during which period the cells stuck to one another to form small aggregates of about lOOµm across. Throughout the experiment, the culture medium was renewed every two days by exchanging 2ml of old medium for 2ml of fresh medium. After four days of culture the cells were exposed to different doses of the phytoestrogens which were dissolved at the appropriate concentration in fresh medium. A 2 ml aliquot of the medium from each well was collected after a further 2 days and stored at -20°C until assayed.
The VTG radioimmunoassay was performed as described previously (Sumpter, 1985).
The phytoestrogens (see fig.la and fig. lb) were This work was suported by an EEC grant attributed to C. Pelissero and J. P. Sumpter.
247
Biochanin A : R = OH; R' = OCH3 HO Uenistein : R = UH; R' = UH
Daidzein : R = H ; R' = OH
Formononetin : R = H ; R' = OCH3 R'
Fig. la. Chenùcal structure of Ûle isoflavonic phytoestrogens used in mis study
synthesized in the "Laboratoire de Cimie Organique et Organometallique, URA 35 CNRS" in Bordeaux according to Pelissero ~ 199la.
H0%00
1 .& .&"<::
0
1 .& OHCoumesirol
H0%0
1 .&1 "<::
.& OH
Equol
Fig. 1 b. Chemical structure of the two other phytoestrogens used in this study.
To assess the viability of our hepatocyte culture system, we tested increasing doses of 17B-estradiol (E2) in order to obtain a dose-response effect on vitellogenin production. One of the dose-response curves obtained is presented in fig. 2.
1000
800
)
i600
s ~ 400
200 0
10 100 1000
Doses of 171>-estradiol (nM)
Fig. 2. Dose-response curve to esirogen. Bars are SD.
It can be seen that a clear dose-response effect was obtained, with increasing doses of 17B-estradiol leading to progressively higher concentrations of VTG in the medium. The sigmoid shape indicates that the action of the estrogen on the hepatocytes occurs via a saturable pathway.
. We ~hen tested the major phytoestrogens as
stunulaung agents on our system. The results obtained are presented in two diagrams. In fig. 3 the potency of three phytoestrogens (biochanin A, daidzein and genistein; see fig. 1) are compared to l 7B-estradiol.
BOO
600 - + - Eslradiol
--..- Biochanin A - - Daiàzein ---.- Ger:Usaein
~ 400
s ~ 200
100 1000 10000 100000
Doses (nM)
Fig. 3. Dose-response curves to 171l-estradiol and thrce phytoestrogens: biochanin A, daid:r.ein, genistein.
Bars are SD.
In fig. 4 the potency of each phytoestrogen with respect to each other is shown; here the VTG production (ng/ml) is presented as a percentage of the VTG concentration induced by 100 nM 17B-estradiol.
&! 120
~ 100
--
BicchaninA§
~
~ ]
~ 'l5
..
Coumestrol
80 Daidzcin
60 Equol
--
Foonooonetin40 Genistein
20
2.S s 10 20 40
Doses(µM)
Fig. 4. Relative action of increasing doses of phytoesirogens on the VTG secretion from cultured hepatocytcs.
Bars are SD.
The relative potency of each phytoestrogen was calculated by the ratio :
[ l 7 B-estradiol] producing 75 ng VTG /ml [phytoestrogen]producing 75 ng VTG/ml
Ali the phytoestrogens were demonstrated to be active on cultured trout hepatocyte with potencies ranging from 1000 to 2000 times Jess than that obtained for 17B-estradiol.
Discussion
The different dose-response curves obtained using our estrogenic subtances demontrate the reliability of the induction of vitellogenin in cultured hepatocytes as a bioassay for estrogenic substances. The sigmoïd shape of the dose-response curves is what is expected, based on the fact that estrogen receptors known to be involved in VTG synthesis (Maitre tl.!!1., 1986; Callard and Callard, 1987) can be saturated with high doses of the test compounds. Also we were able to observe that the height of the plateau obtained with the highest
doses could vary from one cell culture to another depending both on the number of cells in suspension in each well and on the physiological state of our fish when we did the perfusion of the Jiver. These observations agree with the notion that the number of receptors on a target cells varies (Eisenfield tlJ!1
1980). '
Ali the phytoestrogens have been demonstrated to be potent in our jn vitro system. The fact that phytoestrogens are weakly estrogenic is thought to be because of the similarity in chemical structure between phytoestrogens and 17B-estradiol (Duax and Griffins 1985). According to these authors the presence of hydroxyl groups on the two carbons on the opposite ends of the molecules can mimic the disposition of the 17B-estradiol hydroxyl groups. These two hydroxyl groups are known to be crucial in the recognition step when the ~olecule interacts with the estrogen receptor and also m the nature of the action· (agonistic or antagonistic) that they induce on the target cells
(Ka.tzenel~enbog~n et al., 1980). Compared to the data available m the hterature, the range of potencies of the phytoestrogens in trout are similar to the values obtained in mammals (Braden ru!., 1967). However, in marnmals greater differences can be observed from one compound t~ another. The similarity of potencies between the vanous phytoestrogens when tested in
vitro on trout hepatocytes could be due io :
i. a lower specificity of estrogen receptor in fish compared to mammals,
ii. a metabolic degradation of the least potent compounds to more potent metabolites prior to ... interaction with estrogen receptors,
m. to the nature of our system. It must be remembered that in this study we have assessed the potencies of various phytoestrogens in an jn vitro system. It is not really appropriate to compare these potencies to those obtained in vivo (in mammals) because in the latter tests, the phytoestrogens may be catabolyzed enzymatically in the animal, may bind to binding proteins which would alter their half life, may be excreted at different rates, etc.
Conclusion
. This study demonstrated the reliability of our in
YJ.!!Q. system as an estrogenic biological test. It allows us to define an estrogen potency scale for the
p~ytoestr~gen~ with the reservation ~at these might be d1fferent m vivo. The results obtamed raise several questions especially in relation to the activity of phytoestrogens in vivo such as occurs when they are received via the diet
References
Braden A. W. H., N. K. Hart & J. A. Lamberton.
1967. The oestrogenic activity and metabolism of certain isoflavones in sheep. Aust. J. Agric. Res. 18 p. 335-348.
Callard 1. P. & G. Callard. 1987. Sex steroid receptors and non-receptor binding proteins. In: Hormones and Reproduction in Fishes, Amphibians and Reptiles. D. O. Norris & R. E. Jones (Eds) Plenum
249
press. p. 355-384.
Copeland P. A., J. P. Sumpter, T. K. Walker & M.
Croft. 1986. Vitellogenin levels in male and female rainbow trout Salmo gairdneri at various stages of the reproductive cycle. Comp. Biochem. Physiol.
83B p. 487-498.
Duax W. L. & J. F. Griffin. 1985. Structure-activity
~lationsh~ps of estrogenic chemicals.In: Estrogens m the Envrronment. J. A. Mc Lachlan (Ed); Elsevier Pub. p. 15-23.
Eisenfield A. J., Aten R. F. & Dickson R. B. 1980.
Estrogen receptors in the mammalian !iver. In:
Estrogens in the Environment. Mc Lachlan (Ed):
Elsevier Pub. p. 69-96.
Katzenellenbogen J. A., B. S. Katzenellenbogen, T.
Tatee, D. W. Robertson & S. W. Landvatter. 1980.
The chemistry of estrogens and antiestrogens : relatio!1ship~ betwe~n. structure, receptor binding and b1olog1cal acuv1ty. In "Estrogens in the environment." J. A. Mc Lachlan (Ed) Elsevier Pub.
p. 33-51.
Knuckles, B. E., D. de Fremery, and G. O. Kohler.
1976. Coumestrol content of fractions obtained during wet processing of alfalfa. J. Agric. Food.
Chem. 24 p. 1177-1180.
Maitre J. L., L. Mercier, L. Dolo & Y. Valotaire.
1985. Caracterisation de recepteurs à !'oestradiol, induction de vitellogenine et de son mRNA dans le foie de truite arc-en-ciel (Salmo gairdnerii).
Biochimie. 67 p.215-255.
Pelissero, C., B. Bennetau, P. Babin, F. Le Menn &
J. Dunoguès 199la. Estrogenic activity of certain phytoestrogens on vitellogenin synthesis in the Siberian strugeon (Acipenser baeri). J. Steroid.
~iochem. Molec. Biol.38 p. 293-299.
Pehssero C., F. Le Menn & S. Kaushik. 199lb.
Estrogenic effect of Soyabean meal in the cultured Siberian sturgeon Acipenser baeri. Gen. Comp.
Endocrinol. Vol 83. in press.
Setchell K .. D. R. 1985. Naturally occuring non-stero1dal estrogens of dietary origin.In:
Estrogens in the Environment II. J. A. Mc Lachlan (Ed): Elsevier Pub. p 69-85.
Sumpter, J. P. 1985. The purification, radioimmuno-
as~ay and plasma Jevels of vitellogenin from the rambow trout, Salmo gairdneri. In: Trends in Comparative Endocrinology.Lofts B. & Holmes W. H. (Eds) Hong Kong Univ. Press.p. 355-357.
Proceedings
of the
Fourth International Symposium
on the
Reproductive Physiology of Fish
University of East Anglia, Norwich, U. K., 7-12 July 1991
Edited by: A. P. Scott, J. P. Sumpter, D. E. Kime, and M. S. Rolfe
Published by FishSymp 91, Sheffield.
Proceedings of the Founh International Symposium on the Reproductive Physiology ofFish, University of East Anglia, Norwich, U. K., 7-12 July 1991
A. P. Scott, J. P. Sumpter, D. E. Kime and M. S. Rolfe (Editors)
ISBN 0-9517971-0-7
© Copyright 1991, FishSyrnp 91, Department of Animal and Plant Sciences, The University, PO Box 601, Sheffield SlO 2UQ, United Kingdom
No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, mechanical, photocopying, recording or otherwise without prior written permission of the authors.
Printed in the United Kingdom by the University ofEast Anglia Printing Unit
ii
